Bicarbonate-dependent Peroxidase Activity of Human Cu,Zn-Superoxide Dismutase Induces Covalent Aggregation of Protein
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m302051200
ISSN1083-351X
AutoresHao Zhang, Christopher Andrekopoulos, Joy Joseph, Karunakaran Chandran, Hakim Karoui, John P. Crow, Balaraman Kalyanaraman,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoThis study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate ( HCO3−)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3−. HCO3−-dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion ( CO3˙−). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3−-dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3− did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3˙−-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3˙−, forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3−/H2˙O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3−-dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed. This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate ( HCO3−)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3−. HCO3−-dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion ( CO3˙−). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3−-dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3− did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3˙−-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3˙−, forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3−/H2˙O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3−-dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed. In a pair of publications, Hodgson and Fridovich demonstrated that bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD or SOD1) 1The abbreviations used are: SOD, superoxide dismutase; HCO3−, bicarbonate anion; CO3˙−, carbonate radical anion; DMPO, 5,5′-dimethyl-bicarbonate anion; CO3 1-pyrroline-N-oxide; EMPO, 5-ethoxycarbonyl-5′-methyl-1-pyrroline N-oxide; DEPMPO, 5-(dimethoxyphosphoryl)-5′-methyl-1-pyrroline N-oxide; DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid; DBNBS-Trp, DBNBS-tryptophanyl adduct; POBN, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone; ESR, electron spin resonance; DTPA, diethylenetriaminepentaacetic acid; bSOD1, bovine Cu,Zn-SOD; hSOD1WT, wild-type human Cu,Zn-SOD; hSOD1W32F, mutant (Trp-32 to Phe-32) hSOD1-W32F; ALS, amyotrophic lateral sclerosis. exhibits a nonspecific peroxidase activity (1Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5298Google Scholar, 2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Google Scholar). They provided evidence for formation of a potent oxidant in the presence of H2O2 that was ascribed to a copper-bound hydroxyl radical, at the active site of bovine SOD1. Although the rate constant for the reaction between SOD1 and H2O2 is very low (3Liochev S.I. Fridovich I. J. Biol. Chem. 2002; 277: 34674-34678Google Scholar), it was shown that the bovine SOD1 peroxidase activity could oxidize a variety of small molecular weight anionic ligands (azide, nitrite, formate, etc.) that are accessible to the active site (1Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5298Google Scholar, 2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Google Scholar, 4Zhang H. Joseph J. Felix C. Kalyanaraman B. J. Biol. Chem. 2000; 275: 14038-14045Google Scholar, 5Singh R.J. Goss S.P.A. Joseph J. Kalyanaraman B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12912-12917Google Scholar). However, a perplexing aspect of this peroxidase activity was that even larger molecules (e.g. 2,2′-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid, urate) that are not accessible to the active site of SOD1 were also oxidized (6Zhang H. Joseph J. Gurney M. Becker D. Kalyanaraman B. J. Biol. Chem. 2002; 277: 1013-1020Google Scholar). It was later discovered that the bicarbonate anion ( HCO3−) that is present in the buffer was responsible for Cu,Zn-SOD peroxidase-dependent oxidations of substrates in the bulk solution (7Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Google Scholar, 8Kalyanaraman B. Joseph J. Zhang H. Adv. Exp. Med. Biol. 2001; 500: 175-182Google Scholar). It was proposed that the copper-bound oxidant (Cu2+-OH or CuIIIO) could oxidize the HCO3− anion (a physiologically relevant molecule) to the carbonate anion radical (CO3˙−), a potent oxidant that diffuses out of the active site and causes substrate oxidation (7Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Google Scholar, 8Kalyanaraman B. Joseph J. Zhang H. Adv. Exp. Med. Biol. 2001; 500: 175-182Google Scholar). The fact that CO3˙−, and not hydroxyl radical, is the primary oxidant produced by SOD1-mediated peroxidase activity is physiologically significant, because CO3˙− has a much longer half-life (than hydroxyl radical) and can, therefore, diffuse away from the active site and oxidatively modify critical cellular targets. Liochev et al. (7Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Google Scholar) suggested that the nitrone spin trap, 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO), is oxidized and hydrolyzed by CO3˙−. Zhang et al. (6Zhang H. Joseph J. Gurney M. Becker D. Kalyanaraman B. J. Biol. Chem. 2002; 277: 1013-1020Google Scholar) provided the experimental proof for this hypothesis. Using electron spin resonance (ESR) spin trapping in the presence of oxygen-17-labeled water, the investigators showed that the oxygen atom in the DMPO-OH adduct is derived totally from water. Zhang et al. (6Zhang H. Joseph J. Gurney M. Becker D. Kalyanaraman B. J. Biol. Chem. 2002; 277: 1013-1020Google Scholar) also reported that HCO3−-dependent peroxidase activity can be measured using a variety of methods including fluorescence and optical spectroscopy. More recently, Hink et al. (9Hink H.U. Santanam N. Dikalov S. McCann L. Nguyen A.D. Parthasarathy S. Harrison D.G. Fukai T. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1402-1408Google Scholar) demonstrated that an extracellular SOD (SOD3 or ecSOD) enhanced the hydroxylation of a cyclic nitrone spin trap in the presence of H2O2 and HCO3−. In this work, we report that human Cu,Zn-superoxide dismutase (hSOD1WT) also exhibits a HCO3−-dependent peroxidase activity involving the CO3˙− intermediate. However, unlike bovine SOD1, hSOD1WT-mediated HCO3−-dependent peroxidase activity results in protein covalent aggregation. Results from this study show that CO3˙− induces oxidation of a tryptophan residue located on the surface of hSOD1WT that leads to intersubunit covalent bond formation and subsequent aggregation of the protein. Implications of HCO3−-dependent hSOD1WT peroxidase activity in the covalent aggregation of SOD1 associated with amyotrophic lateral sclerosis (ALS) are discussed. Bovine SOD1 was obtained from Roche Diagnostics. Pentammine carbonato complex of Co(III) was synthesized according to the published procedure (10Basolo F. Murmann R.K. Inorg. Syn. 1953; 4: 171-172Google Scholar). Briefly, 30 g of Co(NO3)2·6H2O in 15 ml of water was added to 45 g of ammonium carbonate dissolved in 45 ml of water, followed by the addition of 75 ml of concentrated ammonia. Air was bubbled through the solution for 24 h. The resulting solution was cooled in an ice bath, and the solid product was recrystallized by dissolving it in 55 ml of water at 90 °C and then slowly cooling the solution in an ice bath. Pure crystals were isolated and used in the experiments. Chemicals including glycine, SDS, ammonium persulfate, Coomassie G250 stain, and 40% acrylamide/bis were obtained from Bio-Rad. Sodium bicarbonate, hydrogen peroxide, tryptophan, tyrosine, histidine, phenylalanine, and trizma base were purchased from Sigma. DMPO was also obtained from Sigma, and the colored impurity was removed by treatment with activated charcoal (11Buettner G.R. Oberley L.W. Biochem. Biophys. Res. Commun. 1978; 83: 69-74Google Scholar). POBN, α-phenyl-tert-butyl-N-nitrone, and DBNBS were obtained from Sigma and used as received. EMPO was synthesized according to a previous report (12Zhang H. Joseph J. Vasquez-Vivar J. Karoui H. Nsanzumuhire C. Martásek P. Tordo P. Kalyanaraman B. FEBS Lett. 2000; 473: 58-62Google Scholar). Azulenyl nitrone was a gift from Dr. David Baker (Florida International University, Miami, FL). DEPMPO was obtained from Dr. Paul Tordo (Universite de Provence, Marseille, France). Expression and Purification of SOD1—The wild-type human and W32F mutant Cu/Zn-SOD (Cu2+, 97%; Zn2+, 108%) were expressed and purified as described previously (13Crow J.P. Sampson J.B. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Google Scholar). The SOD1 mutations were created by a two-cycle, polymerase chain reaction-based mutagenesis protocol described by Zhao et al. (14Zhao L.J. Zhang Q.X. Padmanabhan R. Methods Enzymol. 1993; 217: 218-227Google Scholar) using mutagenic primers to introduce the desired mutants and subsequently cloned into a pET-3d expression system (Novagen, Madison, WI). Detailed procedures have been described elsewhere (13Crow J.P. Sampson J.B. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Google Scholar). SOD1 activity was determined by the cytochrome c method (15McCord J.M. Fridovich I. J. Biol. Chem. 1969; 224: 6049-6055Google Scholar). The metallation of the purified SOD1 W32F mutant was determined both by colorimetric PAR assay (13Crow J.P. Sampson J.B. Zhuang Y. Thompson J.A. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Google Scholar) and ESR spectroscopy. 2C. Karunakaran, H. Zhang, J. P. Crow, J. S. Beckman, W. Antholine, and B. Kalyanaraman, unpublished data. Aggregation of SOD1—In general, SOD1 (1 mg/ml) was incubated with H2O2 (1 mm) in a phosphate buffer (100 mm, pH 7.4) containing DTPA (0.1 mm), sodium bicarbonate (25 mm), and other relevant chemical reagents for 45 min. The reaction mixture was then mixed with an equal volume of Laemmli sample buffer containing 5% β-mercaptoethanol and boiled for 6 min. SOD1 (7.5 μg) was loaded onto a 10% SDS-PAGE, and electrophoresis was performed at 4 °C. The protein bands on the SDS-PAGE were visualized by a Coomassie G250 stain and documented using a Multimage Light Cabinet (Alpha Innotech Corp.). The pattern of SOD aggregation was not altered by increasing the amount of sample buffer, boiling time, or β-mercaptoethanol. Bovine or human SOD1 (1 mg/ml) was mixed with the pentammine carbonato complex of Co(III) (4 mm) in a phosphate buffer (100 mm, pH 7.4) containing DTPA (0.1 mm) and other reagents as specified under "Results." The mixture was then irradiated with UV light using an EiMac VIX 300 UV 300 X xenon arc source. The aggregation of SOD1 induced by UV photolysis of the cobalt complex was also assessed by 10% SDS-PAGE. ESR Spin-trapping Analysis—A typical reaction mixture (∼100 μl) for ESR experiments consisted of SOD1 (1 mg/ml), spin traps (25 mm), H2O2 (1 mm), and bicarbonate (25 mm) in a phosphate buffer (100 mm) containing DTPA (0.1 mm). ESR spectra were recorded at room temperature on a Varian E-109 spectrometer operating at 9.5 GHz with a 100-kHz field modulation and equipped with a TE102 cavity. Spectrometer conditions were as follows: modulation amplitude, 1 G value time constant, 0.064 s; scan time, 2 min; and microwave power, 10 milliwatts. Spectral simulations were performed using the WINSIM program (NIEHS, National Institutes of Health, Research Triangle Park, NC). Fluorescence Measurements—Fluorescence experiments were performed on a Shimadzu RF-5301 PC spectrofluorometer (Shimadzu Scientific Instruments Inc.). Spectra were obtained at the indicated excitation and emission wavelengths using 3–5-mm and 10–20-mm slit widths, respectively. Bicarbonate-induced Hydroxylation and Oxidation of Nitrone Spin Traps in the Presence of SOD1/H2O2: Intermediacy of CO3˙−. Radical Anion and Not Hydroxyl Radical—Previously, we reported that HCO3− dramatically enhances hydroxylation and oxidation of the spin trap DMPO to the DMPO-hydroxyl adduct, DMPO-OH (6Zhang H. Joseph J. Gurney M. Becker D. Kalyanaraman B. J. Biol. Chem. 2002; 277: 1013-1020Google Scholar). Using oxygen-17-labeled water, we showed that the oxygen atom in the DMPO-OH adduct formed in the SOD1WT/H2O2/ HCO3−/DMPO system is derived from water and not from H2O2 (6Zhang H. Joseph J. Gurney M. Becker D. Kalyanaraman B. J. Biol. Chem. 2002; 277: 1013-1020Google Scholar). We have now extended this ESR analysis to include other cyclic nitrone traps (EMPO and DEPMPO) and open-chain nitrones (e.g. POBN). The addition of H2O2 (0.1–1.0 mm) to an incubation mixture containing SOD1 (bovine or recombinant human Cu,Zn-SOD; 31.7 μm), spin traps (25 mm of DMPO, EMPO, DEPMPO, or POBN), HCO3− (25 mm), and DTPA (0.1 mm) in a phosphate buffer (100 mm, pH 7.4) yielded the respective ESR spectrum of the corresponding hydroxylated adduct (Fig. 1A). The computer-simulated ESR spectra (shown with dotted lines in Fig. 1A) were obtained using the ESR parameters (Table I). The addition of Me2SO, a frequently used hydroxyl radical scavenger, had no effect on the ESR signal intensity of the hydroxylated adducts (Fig. 1A). In contrast, when Me2SO was added to a mixture containing Fe2+ and hydrogen peroxide (the Fenton system), the ESR spectrum of the hydroxylated adduct was replaced by a methyl radical adduct (Fig. 1B). From these results, it can be concluded categorically that free hydroxyl radicals are not generated in the SOD1/H2O2/ HCO3− system and that ESR can be used to monitor the SOD1/H2O2/ HCO3−-dependent peroxidase activity. These results indicate that Me2SO can be used to differentiate between CO3˙− and ·OH formation. Other scavengers such as ethanol, azide, and formate react with both the hydroxyl radical and the carbonate radical anion and therefore cannot be used to distinguish formation of these species by ESR (Table II).Table IESR parameters of spin adductsSpin adductαNαHβOther couplingsGGGDMPO-OH14.914.9DMPO-CH316.423.4EMPO-OH (23%)14.115.20.85(αHγ)EMPO-OH (77%)14.112.8EMPO-CH315.422.3PBN-OH15.52.7MNP-H14.614.0PBN-CH315.03.3PBN-CO215.94.6POBN-OH15.01.7POBN-CH315.92.7POBN-CO215.63.45DEPMPO-OH14.113.247.3(αP)DEPMPO-CH315.222.347.7(αP)DBNBS-Trp13.60.46(αN)0.64, 0.76, 0.64(αH)0.94(αHmeta) Open table in a new tab Table IIRate constants of hydroxyl radicals and carbonate radicals reacting with various amino acidsHydroxyl Radicalk2 (M-1s-1).OH + AcGly → Product1.7 × 107.OH + Phe → Phenylalanine OH adduct6.9 × 109.OH + His → Product4.8 × 109.OH + TyrOH → Tyrosine OH adduct8.8 × 109.OH + TrpH → Tryptophan OH adduct1.3 × 1010Carbonate RadicalCO3·¯+AcGly→Product<1 × 104CO3·¯+Phe→Product5 × 104CO3·¯+His→Product5.6 × 106CO3·¯+TyrOH→H++CO32−+TyrO•4.5 × 107CO3·¯+TrpH→Trp•+CO32−7.0 × 108 Open table in a new tab In contrast to the cyclic nitrone traps, which form persistent hyroxylated adducts, the hydroxylated adduct of the openchain nitrone, POBN, decomposed to form a secondary radical adduct. Fig. 2 shows the ESR spectra obtained from mixtures containing human SOD1, POBN, H2O2, DTPA, and different concentrations of HCO3− anion in a phosphate buffer. The spectral intensity increased with increasing HCO3− concentrations. The ESR spectra consisted of two adducts corresponding to POBN-OH and the N-tert-butyl hydronitroxide, MNP-H. We propose that POBN-OH decomposed to the aldehyde and MNP-hydroxylamine, which was further oxidized by CO3˙− to the MNP-hydronitroxide. A similar type of radical chemistry has previously been reported for the hydroxyl adduct of α-phenyl-tert-butyl-N-nitrone (16Kotake Y. Janzen E.G. J. Am. Chem. Soc. 1991; 113: 9503-9506Google Scholar). As reported earlier, the proposed mechanism of hydroxylation of nitrones includes a nucleophilic addition of water to either the nitrone-carbonate radical adduct or to the radical cation intermediate (7Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Google Scholar). Independent evidence for the intermediacy of CO3˙− was obtained from photolysis studies using the pentamine carbonato complex of cobalt(III) (7Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1999; 27: 1444-1447Google Scholar). UV photolysis of this cobalt complex has been shown to release CO3˙− (17Copes V.W. Chen S.N. Hoffman M.Z. J. Am. Chem. Soc. 1973; 95: 3116-3121Google Scholar). Irradiation of the pentammine carbonato complex of Co(III) in the presence of nitrone traps yielded ESR spectra of radical adducts that were similar to those obtained from the SOD1/H2O2/ HCO3− system (not shown). Direct ESR detection of the CO3˙− radical formed from mixing concentrated solutions of ONOO– and HCO3− was achieved using a rapid mixing technique, as reported earlier (18Bonini M.G. Radi R. Ferrer-Sueta G. Ferreira A.M. Augusto O. J. Biol. Chem. 1999; 274: 10802-10806Google Scholar). We reproduced this result in our laboratory and observed that the half-life of this species is extremely short (∼6 ms) at physiological pH values (not shown). However, using this rapid mixing technique, we could not detect the carbonate radical anion proposed to be generated in SOD1/H2O2/ HCO3− system (not shown). This is most likely due to a much slower rate of generation of CO3˙− in this system (i.e. rapid decomposition of CO3˙−. combined with a slow rate of formation precluded its accumulation to the levels needed for ESR detection). Bicarbonate Enhances the Aggregation of Human SOD1WT in the Presence of H2O2—The addition of hSODWT to solutions containing H2O2 (1 mm) and DTPA (0.1 mm) in a phosphate buffer (100 mm, pH 7.4) in the presence of varying concentrations of HCO3− (0–25 mm) caused a concentration-dependent increase in the formation of a hSOD1WT dimer (42–43 kDa) (Fig. 3A). Fig. 3B shows the time-dependent formation of the hSOD1WT dimer from incubations containing hSOD1WT, H2O2, and 25 mm HCO3− anion in a phosphate buffer. The existence of an SOD1 dimer under reducing SDS-PAGE conditions indicates that the subunits are covalently cross-linked via a nondisulfide type linkage. Formation of covalently linked higher molecular weight species (equivalent to dimers of 18-kDa SOD1 subunits) was initially noticeable after 15 min, and with prolonged incubation (60–120 min), fragmentation occurred. Fig. 3, C and D, depicts covalent cross-linking of hSOD1WT as a function of increasing H2O2 and hSOD1-WT concentrations. We then showed that UV photolysis of a cobalt complex that generates authentic CO3˙− radicals in the presence of hSOD1WT caused its cross-linking (Fig. 3E). There was no apparent dimer formation cross-linking in the dark from incubations containing hSOD1WT and the cobalt complex (Fig. 3E). These results provide additional evidence for CO3˙−-induced aggregation and covalent cross-linking of hSOD1WT. Inhibition of hSOD1 Aggregation by Nitrone Spin Traps— The addition of the nitrone spin traps DMPO (25 mm), α-phenyl-tert-butyl-N-nitrone (25 mm), and azulenyl nitrone (4 mm) to an incubation containing hSODWT, H2O2, DTPA, and HCO3− in a phosphate buffer blocked apparent dimer formation, as shown in Fig. 4A. Fig. 4B shows the densitometric analysis of the dimer band obtained in the presence of spin traps. Nitrone spin traps also inhibited UV/cobalt complex-induced covalent dimerization of hSOD1WT (Fig. 4C). Me2SO, a well known trap for hydroxyl radicals, had no effect on hSOD1WT dimer formation (Fig. 4D). These findings are consistent with the notion that trapping of CO3˙− by nitrones inhibits hSOD1WT covalent dimerization. The Effect of Amino Acids on Aggregation of SOD1 in a Human SODWT/H2O2/ HCO3− System—As shown earlier, evidence for enhanced formation of the hSOD1WT covalent dimer was noticeable in incubations containing hSOD1WT, H2O2, and HCO3− (Fig. 5A, lane p). Inclusion of glycine or phenylalanine had no effect on hSOD1WT covalent dimer formation induced in the hSOD1WT/H2O2/ HCO3− system. Both tryptophan and tyrosine totally inhibited covalent dimer formation (Fig. 5A). Similar results were obtained during cobalt complex-photosensitized hSOD1WT covalent aggregation (Fig. 5C). These results suggest that tryptophan and tyrosine scavenged the oxidant (i.e. CO3˙−) responsible for hSOD1WT covalent aggregation. We used DMPO to measure the HCO3−-dependent hSOD1WT peroxidase activity. The DMPO-OH signal (Fig. 5B) was markedly inhibited in the presence of tryptophan and tyrosine (1 mm). At these concentrations, this signal intensity was not affected by phenylalanine and glycine. These data are consistent with the rapid scavenging of the carbonate anion radical by tryptophan and tyrosine (19Chen S.N. Hoffman M.Z. Radiat. Res. 1973; 56: 40-47Google Scholar). From these results, we conclude that either a tryptophanyl or tyrosyl residue present in hSOD1WT is the proximal site of interaction with CO3˙− generated during HCO3−-dependent hSOD1WT peroxidase activity. Spin Trapping of the Bicarbonate-mediated Protein Radical Formed in the Human SOD1/H2O2System—An ESR spectrum that is characteristic of a strongly immobilized nitroxide adduct was detected from incubations containing hSOD1WT, H2O2, HCO3−, and DBNBS trap (Fig. 6A, top). When the HCO3− anion was excluded, no spectrum was obtained (Fig. 6A, middle). Under the same experimental conditions, bovine SOD1 did not form a similar nitroxide adduct (Fig. 6A, bottom). Following ultrafiltration, treatment of the nitroxide adduct (Fig. 6B, top) with the Pronase enzyme that cleaved the high molecular weight nitroxide into a low molecular weight nitroxide yielded an isotropic three-line ESR spectrum (Fig. 6B, middle) with a hyperfine coupling constant of 13.6 G. Upon expansion of the center line of the ESR spectrum (Fig. 6B, middle), superhyperfine couplings were resolved (Fig. 6B, bottom). This spectrum was simulated (dotted line in Fig. 6B, bottom) using the following parameters (αN, 0.37 G; αH1, 0.13 G; αH2, 0.95 G; αH3, 0.58 G; αHm (2Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Google Scholar), 0.92 G). In the presence of tryptophan, the incubation mixture containing bovine SOD1, H2O2, HCO3−, and DBNBS yielded an intense isotropic three-line ESR spectrum (αN = 13.6 G) (Fig. 6C, top). Without HCO3−, no ESR spectrum was obtained (Fig. 6C, middle). Upon expansion of the center line of the ESR spectrum (Fig. 6C, top) using a lower modulation amplitude, superhyperfine couplings could be resolved (Fig. 6C, bottom). This spectrum was simulated using contributions from a nitrogen atom, three nonequivalent protons, and the two meta-protons present in DBNBS, as reported previously (20Gunther M.R. Kelman D.J. Corbett J.T. Mason R.P. J. Biol. Chem. 1995; 270: 16075-16081Google Scholar) (dotted line in Fig. 6C, bottom) (Table I). The structure of the adduct was assigned to the DBNBS-tryptophanyl adduct (DBNBS-Trp). Based on these results, the immobilized nitroxide spectrum (Fig. 6A, top) is attributed to trapping of a radical formed from the tryptophan residue in hSOD1WT. Concomitant with the trapping of the radical formed from the tryptophan residue, DBNBS markedly diminished HCO3−/hSOD1WT peroxidase-dependent covalent dimer formation from hSOD1WT (Fig. 6D). The Effect of Substituting Phenylalanine for Tryptophan on Bicarbonate-mediated Hydroxylation and Oxidation Reactions in the hSOD1W32F/H2O2System—The wild-type human SOD1 has a single tryptophan residue (Trp-32) located on the surface of the protein, as shown in the ribbon diagram model (Fig. 7A). To assess the role of tryptophan in hSOD1WT/H2O2/ HCO3−-mediated oxidation and aggregation reactions, a site-directed mutant of hSOD1, containing Phe in place of Trp-32, was produced (Fig. 7B). The fluorescence spectra of hSOD1WT and hSODW32F confirmed the loss of tryptophan fluorescence upon substitution with phenylalanine (Fig. 7, dotted line). The copper ESR spectra of hSOD1WT and hSOD1W32F were identical, demonstrating no change at the active copper site (not shown). The next step was to compare the ESR spectra of the DBNBS adducts obtained from incubation mixtures containing either hSOD1WT or hSOD1W32F, DBNBS trap, H2O2, and HCO3− in a phosphate buffer (100 mm, pH 7.4) containing 0.1 mm DTPA. An intense ESR spectrum of a protein-derived radical was detected from the hSOD1WT (Fig. 8A). No immobilized ESR spectrum was detected from the hSOD1W32F- HCO3−-mediated reaction (Fig. 8C). The HCO3−-dependent peroxidase activity of hSOD1WT and hSOD1W32F was monitored by DMPO hydroxylation to DMPO-OH (Fig. 8, B and D). The lack of Trp-32 in hSOD1W32F actually enhanced hSOD1W32F/H2O2/ HCO3−-dependent DMPO-OH formation (Fig. 8D), as compared with hSOD1WT/H2O2/ HCO3−/DMPO (Fig. 8B). This difference may be explained in terms of the reduced availability of the CO3˙− radical (due to increased stoichiometric scavenging of CO3˙− by Trp-32) for DMPO hydroxylation by hSOD1WT, H2O2, and HCO3−. This was further verified by using free tryptophan as a substrate for SOD1/bicarbonate-mediated peroxidase activity. The spin trap DBNBS was used to trap the tryptophan-derived carbon-centered radical. The ESR spectra of DBNBS adducts obtained from incubations containing hSOD1WT or hSODW32F, DBNBS, H2O2, and HCO3− in a phosphate buffer containing DTPA are shown in Fig. 8, A and C. In the presence of 50 μm free tryptophan, an ESR spectrum due to the DBNBS-Trp adduct was detected from hSOD1W32F/H2O2/ HCO3−/DBNBS system (not shown), and at 1 mm free tryptophan, the intensity of the spectrum was higher (not shown). At a lower concentration of free Trp, spectra from both DBNBS-hSOD1WT and DB-NBS-Trp were detected; however, at 1 mm free Trp, the ESR spectrum was solely due to the DBNBS-Trp adduct and was almost the same as that observed from hSOD1W32F (not shown). These results unequivocally demonstrate that hSOD1WT and hSOD1W32F exhibit the same extent of bicarbonate-mediated peroxidase activity, and the lack of ESR spectrum from hSOD1W32F suggests that CO3˙− reacts with the tryptophan residue to form a tryptophan-derived carbon-centered radical that was trapped by DBNBS. It was then of interest to find out whether substituting Trp-32 with Phe-32 has any effect on the aggregation and covalent cross-linking of the protein. We compared the covalent aggregations of hSOD1WT and hSOD1W32F in the presence of H2O2 and HCO3−. Fig. 8E shows that covalent dimerization of hSOD1W32F did not occur under the same experimental conditions (Fig. 8A) that resulted in covalent dimerization of hSOD1WT. Clearly, the Trp residue at position 32 plays a crucial role in the oxidation, aggregation, and covalent cross-linking of hSOD1WT caused by HCO3−-mediated peroxidase activity. The Effect of Molecular Oxygen on the Fluorescence Spectra of Tryptophan-derived Oxidation Products—The UV-visible and fluorescence spectral changes in the oxidation products of tryptophan in a bovine SOD1/H2O2/ HCO3− system are shown in Fig. 9. Bicarbonate accelerated the rate of oxidation of tryptophan in the presence of SOD1 and H2O2 (Fig. 9, A (middle) and B (left)). Similar spectral changes due to oxidation of Trp-32 residue were observed in the hSOD1WT/H2O2/ HCO3− system (Fig. 9, A (right) and B (middle)). Fluorescence spectra (Fig. 9B, right) revealed at least three different products that are formed during HCO3−/H2O2-dependent oxidation of Trp-32 in hSOD1WT. The fluorescent intensities of two products (Fig. 10, A and B) were monitored under aerobic and anerobic conditions in the hSOD1WT/H2O2/ HCO3− system. Product formation was considerably enhanced in air. Bicarbonate/H2O2-dependent dimerization of hSOD1WT was enhanced in air and diminished in N2 (Fig. 10C). These
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